Metabolomics-based profiling for quality assessment and revealing the impact of drying of Turmeric (Curcuma longa L.)

Turmeric, the rhizomes of Curcuma longa L., is one of the top selling spices, food preservatives, and food colorants. In addition, it exhibits health promoting benefits owing to its unique phytochemical composition. Nevertheless, it is commonly subjected to heat drying, hence, the dried powder is the most used form and can easily be adulterated with allied species. Therefore, our research aimed to profile the phytochemical composition and investigate the impact of drying of turmeric. Extraction and fractionation followed by LC- and GC–MS analysis resulted in the identification of a total of 161 metabolites belonged to various phytochemical classes. Moreover, multivariate data analysis identified curcuminoids, terpecurcumins, and organic acids as potential markers for drying. Based on the applied analytical techniques in combination with chemometrics, these investigations have succeeded to provide good coverage of the metabolome of turmeric in both fresh and dried forms.

www.nature.com/scientificreports/ affect the organoleptic properties of the herb, i.e., the fresh form possesses an aromatic and spicy fragrance, yet a distinctive medicinal aroma results upon drying 13 . It is also noteworthy to find that previous reports investigated the effect of various drying methods with focusing on their optimization related to product quality. For instance, Hirun, et al. investigated the microwave-vacuum drying approach in terms of microwave power (2400-4000 W) and drying times (10-30 min), where they showed significant effects on the end product's colour via inhibition of polyphenol oxidase, moisture content, ash, phenolic content, curcuminoid content, and antioxidant activity as well, especially at higher power and longer drying times 5 . Asides, the effect of mechanical drying air employing tray drier was studied in terms of drying air temperatures (45-65 °C) and velocities (1-3 m/sec) on the powder quality 14 . Recently, hot-air dryer coupled with a simulated solar radiation was applied for turmeric drying. The results showed that the dried products possessed intensive orange color. Additionally, curcumin, demethoxycurcumin, and total curcuminoids were influenced, where the lowest curcumin content was detected at 40 °C under PMMA 15 . Adulteration of turmeric powder, in contrast with the whole fresh or dried rhizome, has been frequently reported including the mixing with C. zedoaria (white turmeric) and azo compounds, i.e., metanil yellow and Sudan Red G 4,7 . Detection of these potential contaminants and powder authentication were based on curcumin and dye determination using high-performance liquid chromatography (HPLC), high performance capillary electrophoresis, HPLC-electrospray ionization tandem mass spectrometry (HPLC/ESI-MS), UV-vis spectroscopy coupled with multivariate analysis, and recently with Fourier Transfer-Infrared (FT-IR) and FT-Raman spectroscopic systems 7,16 . In addition, a DNA barcoding method was developed for the detection of plantbased adulterants in turmeric powder 17 . Besides these approaches, the concept of using chemometrics tools, i.e., multivariate data analysis including principal component analysis (PCA), has recently shown to assess quality, discrimination, and authenticate herbal spices based on volatiles, polyphenolics, and curcuminoids profiles 18 .
Drying could be achieved by different methods for example freeze drying, sun drying, microwave drying, oven-drying, among others 19,20 . Various enzymatic and non-enzymatic reactions occur during the drying process which lead to great variation in the bioactivity and chemical composition of the primary and specialized metabolites 21,22 . Metabolomics allows the high-throughput analysis of all metabolites, which is valuable in detection of the possible biotransformation between the primary and secondary metabolites during the food processing 19,21,23 . Changes in the chemical compositions due to the processing methods will lead to several changes in the biological activity of the plant, especially if the beneficial effects are not attributed to one single component, but due to several the constituents found in the plants 20,[24][25][26][27][28] . Consequently, the proper method should be designated to optimize the yield of the target metabolites.
Generally, amino acids in their free form are known as the major taste-active compounds in the foodstuffs. The free amino acid compositions were reported to be altered by the different drying process. Additionally, the drying at high temperatures affected the nutrient content, color and physico-chemical properties, sensory characteristics, including aroma, flavor, and texture. Potentially decreased the quality and market value of the functional ingredients by the food industry.
Given the importance of turmeric and its richness in phytochemical composition, a comprehensive analysis of turmeric rhizome is critically required to ensure both safety and quality. In addition, assessment of the impact of drying on its chemical profile is necessary. Therefore, the current research comprehensively characterized the primary and specialized metabolites of fresh and dried turmeric via various modern sensitive chromatographic techniques determining the appropriate form either for nutrition or medicine. In addition, chemometrics tools were applied for the identification of potential markers that can be used for authentication and discrimination between fresh and dried samples.

Results and discussion
In order to cover the diverse and wide spectrum of metabolites in either fresh or dried turmeric rhizomes, a comprehensive extraction protocol was conducted. In addition, various hyphenated analytical methods were performed to identify potential markers in the complex matrix of metabolites and to investigate the effect of drying (Fig. 1).
The total ion chromatograms (TIC) of turmeric are shown in Fig Table S1. Their relative abundances were affected by drying, where the results demonstrated that curcuminoids abundance increased after herbal drying including all members, in accordance with previous reports 29 , except curcumalongin A and curcumin dimer (Table S1 and Fig. 3). In addition, demethoxy curcumin and bisdemthoxycurcumin were the most abundant metabolites among all members of curcuminoids.
Structural annotation of curcuminoids (curcumin, desmethoxycurcumin, bisdesmethoxycurcumin) (Suppl. Sesquiterpenoids. Eleven sesquiterpenoids metabolites were detected (Table S1). They were detected mostly in the non-polar MTBE fraction in addition to few numbers in polar MeOH/H 2 O analyzed by UPLC-C 8 -FT-MS/MS and UPLC-C 18 -FT-MS/MS, respectively. The relative abundances of sesquiterpenoids showed clear differences between fresh and dried samples, where all of them were more abundant in fresh than dried samples and likely to be affected by drying process. For instance, the relative abundances of ar-turmerone and furanodiene were decreased approximately to half upon drying, Table S1 and Fig. 3.
Particularly, the bisabolane constituents, i.e., xanthorrhizol followed by α-turmerone and ar-turmerone were the most abundant sesquiterpenoids. These results agreed with previous literature confirming that, for example, α-turmerone, and ar-turmerone, are key taxonomic markers for turmeric 31 . However, xanthorrhizol is a unique constituent of C. xanthorrhiza Roxb., commonly known as Java turmeric, and a wide variety of biological properties have been reported for this metabolite 29 . Hence, detection of xanthorrhizol as a major constituent of either fresh or dried C. longa has been reported for the first time.
Terpecurcumins. Terpecurcumins are bioactive metabolites isolated from the rhizomes of turmeric. They are synthesized through the hybridization of curcuminoids and bisabolanes 32 . Various terpecurcumins were identified mostly in the non-polar fraction of turmeric and analyzed by UPLC-C 8 -FT-MS/MS. Results revealed that tercurcumin Q and terpecurcumin R were the most abundant members of this class. In addition, their abundances were variable between dry and fresh samples. For example, most of terpecurcumins were more abundant in dried samples, similar to curcuminoids, as terpecurcumin Q, R, and D. However, others as terpecurcumin S, A/B/C, and J were much more in fresh analogues, Table S1 and Fig. 3. The research has thus distinguished between fresh and dried turmeric regarding terpecurcumins for the first time.  Table S1 and Fig. 3. Among the identified lipid components were DGDGs (13), GlcADG (six), MGDG (six), SQDGs (three), TAG (10), and phospholipids (seven). Their abundances were mostly relatively high in fresh samples than dried. Moreover, seven fatty acids were identified including essential unsaturated types as 18:3, 18:1, and 16:1. Their relative abundances were also generally higher in fresh samples. Essential fatty acids are considered key nutrients that affect growth, development and nutrition-related chronic disease 33 . Other miscellaneous compounds. Amino acids, sugars, and organic acids were detected in polar fraction following derivatization and analysis by GC-TOF-MS. As shown in Suppl. Fig. S2, pyroglutamate, alanine, www.nature.com/scientificreports/ serine, asparagine, aspartate, and glycine were the most abundant among amino acids. These metabolites were shown to decrease upon drying. In addition, the abundances of sugars were essentially similar between fresh and dried samples, except in case of raffinose which highly increased after drying. Sucrose and myo-inositol were the most abundant and additionally were relatively more abundant in fresh samples. Such abundant sugars may contribute to the sweet taste of the fresh rhizomes, especially given the lower abundances of curcuminoids in the fresh rhizomes. myo-Inositol has purported health promoting effects, where it is suggested to provide promising effects against different diseases including diabetes and cancer 34 . The levels of myo-inositol were not significantly affected by drying, indicating that the dried form is also effective. Moreover, the content of the trisaccharide raffinose was nearly double in dried samples indicating that it may be a byproduct of a certain decomposition reaction induced by heating. Interestingly in plants, its presence is always associated with protection against oxidative damage 35 .
Furthermore, the level of organic acids and the effect of drying on their abundances were investigated. Citric acid was the most abundant in both fresh and dried samples. Yet, its abundance was higher in fresh rhizomes. Other major acids were 4-amino-butanoic acid, fumaric acid, and gluconic acid. However, effect of drying on the levels of these compounds was unclear and variable. While 4-amino-butanoic acid, gluconic acid, and 1-dehydro-ascorbic acid were more in fresh samples, others as fumaric acid, glyceric, and pyruvic acids were in dried samples.
Also, β-citraurol and β-citraurin are among apocarotenoids that are derived from carotenoids through oxidative cleavage, and hence may affect the powder color. The effect of drying was relatively different, i.e., while β-citraurol was more abundant in fresh samples, β-citraurin was in dried counterparts. Flavonoids were also among the identified compounds in turmeric, which have not been well investigated in previous literature. The current research could identify two flavonoid glycosides, i.e., hesperidin and quercetin di-rhamnoside, in the MeOH/H 2 O fraction. The results showed that they were richer in fresh samples, Suppl. Fig. S2.
Interestingly, the research revealed the presence of compounds in turmeric for the first time. Examples included drovomifoliol-O-glucopyranoside, oleuropeoylsucrose, and corchoionoside B which were previously detected in the Egyptian Murcott mandarin waste 36 , myrtaceous species 37 , and Vietnamese Corchorus olitorius L. 38 , respectively. The presence of these compounds would increase the medicinal interest and the importance of turmeric.

Multivariate data analysis of turmeric samples. A principal component analysis (PCA) score plot
revealed a clear segregation of the dried from fresh samples of turmeric mainly across PC1 representing 85.4% of the total variance (Fig. 4A). In addition, Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) score plot confirmed the PCA findings, Fig. 4B. The OPLS-DA S-plot demonstrated that raffinose threose, glycolic, fumaric, pyruvic acids, and monomethyl curcumin potentially marked dried samples, while metabolites such as amino acids and sesquiterpenoids were more associated with fresh rhizomes, Fig. 4C. Hierarchical cluster analysis (HCA) revealed separation of fresh and dry samples to two different clusters reflecting the metabolic composition (Fig. 4D).
In addition, a Volcano plot was traced to show the impact of drying. The results showed that at p < 0.05, the relative abundances of 51 metabolites were decreased upon drying (negative side), while 23 increased (positive side) and 87 were not significantly affected. Examples of metabolites that decreased by drying mainly belonged to amino acids such as glutamine, tryptophan, serine and homoserine, as well as glutathione (oxidized form). In contrast, threose, mono demethylcurcumin, and glycolic and fumaric acids were examples of metabolites that were enriched upon drying in turmeric rhizomes, Fig. 5A.
A variable importance in projection (VIP) plot displayed the top 20 most important metabolite features identified by OPLS-DA (Fig. 5B). For instance, amino acids and phospholipids can be recognized as markers for fresh turmeric, while terpecurcumins and tetrahydrodemethoxycurcumin are for dried counterparts. Most of the previously published articles have focused on quantitative and qualitative profiling of curcuminoids and essential oils contents of turmeric oleoresin. However, the current research has succeeded to profile the phytochemical composition of turmeric more comprehensively following application of an efficient metabolomics approach. The presented workflow is a powerful tool for the quality assessment, authentication and discrimination between different forms of food or herbal medicine.

Conclusion.
A relatively comprehensive phytochemical analysis combined with multivariate analyses of food spices attracted considerable attention as a potential quality assessment tool. Since drying is still the most applicable and ancient method for herb and food preservation, the effect of drying has been studied regarding the nutritional, organoleptic characteristics, and medicinal use determining the most suitable form for each use. The impact of heat drying was investigated on turmeric rhizomes within metabolomics-based study for the first time. Intriguingly, most classes of lipids, sesquiterpenoids, flavonoid glycosides, and amino acids were more abundant in fresh samples, while curcuminoids and terpecurcumins increased by drying. Hence, the nutritional value and aromatic property may be decreased by drying, in contrast to the medicinal applications. This point raised the questions of how to optimize the drying process getting both nutritional and full aromatic values of turmeric beside the medicinal benefits. Preparation and drying of samples. Firstly, the outer cork tissues were removed carefully with a knife.

Material and methods
Then, the peeled rhizomes were cut into small pieces (1 mm in length) of the same thickness. In parallel, a portion of the peeled turmeric rhizomes was lab-dried by subjecting to an oven adjusted at 40 °C for 1.5 days [39][40][41] . The selection of drying time and temperature was based on the preliminary trials for optimum drying without burning or inducing severe changes in the color of the rhizomes as well as the available reported literature [42][43][44] . Previous studies found that the turmeric rhizomes that were subjected to oven-drying at 60-100 °C showed changes in total phenolic content, colour value as well as polyphenol oxidase activity 43 . The time of drying was  Sample preparation for analysis. Extraction protocol and phase separation. Frozen turmeric samples were homogenized and then subjected to our previously developed extraction and fractionation protocol 45 . In brief, 10 mg of each replicates, calculated on dry weight basis, were incubated in a micro-centrifugation tube with pre-cooled (1 mL, −20 °C) methyl tert-butyl ether (MTBE):methanol (MeOH) (3:1, v/v). Then, a phase-

Preparation of samples for MS-based analysis.
For analysis of the non-polar fraction, aliquots (500 µL) from the MTBE layer were dried for 3 h in a vacuum centrifuge. The dried pellets were resuspended in 400 µL acetonitrile:isopropanol (70:30) before they were subjected to UPLC-C 8 -FT-MS/MS analysis. Semi-polar to polar metabolites were analyzed via two independent aliquots (300 and 150 µL) from MeOH/H 2 O layer. Both aliquots were dried in a vacuum centrifuge for 6 h. Then, the dried pellets, resulted from the 300 µL aliquots, were resuspended in 200 µL 50% aqueous MeOH before they were subjected to UPLC-C 18 -FT-MS/MS analysis 46 . The other dried pellets, resulted from of 200 µL aliquots, were derivatized before they were subjected to GC-TOF-MS analysis 47 .
Chromatography coupled with mass spectrometry instrumentation. The UPLC-FT-MS/MS consisted of a Reversed Phase (RP) column (100 × 2.1 mm, 1.7 μm diameter particles, Waters®) in a Waters Acquity UPLC system (Waters®, Manchester, UK). The chromatography unit was coupled to a mass spectrometer able to work in both ionization modes, i.e., positive and negative, using a heated electrospray ionization (HESI) source together with Orbitrap-type MS (Exactive, Thermo-Fisher®, Bremen, Germany) 47 . Additionally, GC-TOF-MS was performed for derivatized samples with MSTFA 48 .
Data analysis. The LC or GC datasets were imported into Microsoft excel (Excel 2010, Microsoft®, Redmond, USA). Only significant peaks, i.e., with at least five-fold higher intensity in comparison to the blank peaks, were further considered for data analysis. Metabolite data obtained were then normalized by weight and internal standard normalization. All statistical analyses were performed using the algorithms embedded within Microsoft excel. In addition, multivariate data analysis (MVDA) was conducted using Metaboanalyst 3.0 software 49 .